It seems
plausible that the presence and/or absence of calcium ions on specific areas
of ion pores/channels results in a change of the protein’s shape. Calcium
may affect the distribution of electrons on the protein and alter the
atomic/intra-molecular bond angles. This could modify the structural
conformation of the pore/channel proteins, creating differences in membrane
permeability to specific ions.]

Cell
membranes, which are vital to the maintenance of intra and
extracellular ion concentrations, appear to be
important ion permeability barriers in fish. Generally, a cell from any
vertebrate is bathed in extracellular fluid
(blood and interstitial fluids). The fluid found within the cell
(intracellular fluid) has a distinct ionic composition, different from
extracellular fluid. By maintaining this ionic
disparity, cells are able to maintain internal fluid pressure and hence
their shape. Additionally, a membrane potential (an electrical voltage
gradient) is generated by maintaining the difference in ionic composition as
well as differences in charged components across the cell membrane. This
occurs as a result of the semi-permeable nature (differential permeability
to select ions) of cell membranes. The ionic gradient is thought to be
generated and maintained by energy dependent processes. Many of the
regulated ions have important functions for enzymatic reactions, the
generation and conductance of electrical impulses and the maintenance of
cell integrity.

The two
ions which are most conspicuously regulated are sodium and potassium. The
cell membrane is 100 times more permeable to potassium than sodium. The
concentration of sodium is highest in extracellular
fluid while potassium is highest in intracellular fluid. Cell membranes
become less permeable to molecules as they increase in size as well as
charge or polarity. The cell membrane behaves as if it had numerous pores
traversing it. These hypothesized pores appear to have a diameter of 8.0
angstroms and could account for the differences in membrane permeability to
sodium and potassium (Solomon, 1960; Guyton, 1971). More recent work has
described and elucidated the presence of specific cellular pores or channels
from excitable tissues (Matsuda and Noma, 1984;
Moczydlowski et al., 1985). It is postulated
that the membrane pore is lined or gated with positively charged prosthetic
groups. Further, it is theorized that the prosthetic groups are divalent
cations; calcium, in particular. These ions are
attached presumably to binding sites along the surface of the pore (Frankenhaeuser
and Hodgkin, 1957; Solomon, 1960; Guyton, 1971;
Moczydlowski et al., 1985).

An
element in its ionic form has an electrical charge whose field lines
approximate a sphere, at the center of which is located the atom. It is
believed the charged fields of divalent calcium ions allow only certain
molecules to pass through the membrane pore. The pore itself restricts the
size of particles passing through it. Positively charged calcium ions
lining the pore restrict the passage of other positively charged ions on the
basis of the magnitude of their charged sphere. Sodium has a greater sphere
of positive charge than potassium and should pass through the cell membrane
(pore) less readily than potassium. This theory is compatible with the
presence of higher concentrations of sodium and lower concentrations of
potassium in extracellular fluids with the
converse being true for intracellular fluids. Ostensibly, the pore (passive
mechanism) coupled with energy dependent ion pumps (active mechanism)
sustain this gradient.

A
pertinent model for studying ion fluxes across cell membranes is the nerve
cell. The nerve cell generates, conducts and transmits electrical impulses
to the tissue that it innervates by creating a wave of membrane
depolarization. This is caused by a rapid but transient change in the
permeability of the cell membrane to both sodium and potassium ions.
Depolarization is caused by a rapid influx of sodium ions into the nerve
cell which disrupts the charge gradient across the membrane. This
depolarization starts at one point on the cell membrane and spreads over the
entire surface of the nerve, apparently by exciting (depolarizing) all areas
of the cell membrane directly contiguous to the initial point of
depolarization. It has been hypothesized (Frankenhaeuser
and Hodgkin, 1957; Guyton, 1971) that a sudden yet temporary removal of the
charged calcium that lines the membrane pore allows sodium to more readily
penetrate or move through the membrane. This could possibly occur in
response to the release of nerve transmitters, hormones or other unknown
chemical substances.

Calcium
is then restored preventing further sodium influx. When this occurs, a
transient efflux of potassium from the intracellular fluid to the outer
surface of the cell membrane restores the original surface charge, thus
repolarizing the membrane. It is further
recognized that once a point area of membrane is depolarized, depolarization
continues spontaneously. The wave of depolarization is self propagating
(Guyton, 1971). It is assumed that the mechanism of depolarization (the
increase in sodium influx) is still the same. The wave of increased sodium
permeability would result from altering the binding affinity for calcium in
the membrane pore. This could occur as a result of the changed charge on
the surface of depolarized membrane contiguous to membrane that has not been
depolarized. The change in charge of depolarized membrane could alter the
calcium binding affinity of the pores in stable membrane by affecting the
charge of the calcium binding sites (possibly pore proteins). Nerve
transmitters and hormones may effect depolarization by the same mechanism.
Ion fluxes across cell membranes, as described above, appear to be transient
and not directly dependent on energy (ATP).

It has
been recognized that the intensity of stimulus necessary to initiate sodium
influx can be reduced by lowering the concentration of calcium in the
extracellular fluid (Frankenhaeuser
and Hodgkin, 1957; Guyton, 1971). Presumably, this occurs in response to an
altered binding affinity for calcium in the membrane pores. These
observations are consistent with the principles of cofactor-enzyme
interactions as defined by Michaelis-Menten
enzyme kinetics (Lehninger, 1975). The affinity
or rate of cofactor binding (calcium in this example) is dependent upon the
concentration or availability of that component in the reaction medium (extracellular
fluid bathing the nerve cell membrane). Low calcium in the
extracellular fluid would result in incomplete
saturation of the calcium binding sites along the membrane pore, reducing
the stimulus (force) necessary to dislodge them in the process of
depolarization. If calcium concentrations are sufficiently low in the
extracellular fluid, spontaneous sodium influx
will occur (Frankenhaeuser and Hodgkin, 1957;
Guyton, 1971). An understanding of this hypothetical mechanism simplifies
the explanation of the results of this dissertation and provides a parallel
analogy for comparison with the effects of environmental calcium on red drum
performance.

The
extracellular fluids (blood and interstitial
fluids) of the red drum or any fish come into close contact with the
environment by way of the body surfaces and the gills. The
extracellular fluids of
teleosts have a unique and relatively stable ionic composition. In
general, most vertebrates are similar. Human
extracellular fluid has sodium, potassium and calcium at
concentrations of 3264, 196 and 100 mg/1 respectively (Guyton, 1971). When
compared, a wide variety of teleosts show
similar ionic concentrations in their extracellular
fluids (Holmes and Donaldson, 1969).

The
environment, whether fresh or saltwater, has unique yet different ionic
characteristics from the extracellular fluids of
teleosts. Most marine waters (35 g/1 TDS) have
much higher concentrations of sodium, potassium and calcium at 10685, 396
and 410 mg/1 (Gross, 1977), respectively, than the
extracellular fluids of teleosts (e.g.
red drum). Conversely, the concentrations of these ions in freshwater
(Boyd, 1979) are typically well below those found in the
extracellular fluids of
teleosts. Therefore, the differences in ionic composition of
teleostextracellular
fluid and that of the environment generate an ionic gradient across the
membranes (semi-permeable) of teleost surface
epithelia.

The
surface epithelia of gills and body surfaces are protected from direct
interaction with the environment by mucous and intercellular junctions.
Fish mucous appears to have calcium binding properties (ChartierBaraduc, 1973). Mucous is a glycoprotein and
could serve as a calcium chelating agent retarding ion loss from epithelial
cells as a charged surface coat or barrier. Intercellular junctions are
specialized areas of attachment between epithelial cells preventing the loss
of ions and fluids from the interstitial area beneath (Bloom and Fawcett,
1975). It has been postulated that cell membranes and intercellular
junctions are dependent on calcium for normal function (Loewenstein,
1966; Guyton, 1971; Bloom and Fawcett, 1975). This dependence may relate to
the routes (perhaps pores or channels) of passive and active ion movements
(Guyton, 1971; Fromter and Diamond, 1972, Claude
and Goodenough, 1973;
Sardet et al., 1979; Sardet, 1980;
Nonnotte et al., 1982). While some ion loss or
gain occurs as a result of leakiness, it is assumed that energy dependent
processes can compensate.

Molecules
or ions diffuse from areas of high concentration to areas of low
concentration until they are equally distributed. This is true for each
specific component of any solution. It is of interest for this discussion
that sodium, potassium and calcium concentrations in sea water are higher
than those of fish extracellular fluid while
those of fresh water are lower. In sea water, the tendency is for sodium,
potassium and calcium to diffuse into fish
extracellular fluid. In fresh water, the reverse would occur. It is
assumed that what diffusion does occur, does so through ion pores in
epithelial cell membranes and intercellular junctions (gills and body
surfaces). Permeability barriers (ion pores and mucous) and energy
dependent ion pumps prevent the ionic composition of fish
extracellular fluid from equilibrating with that
of the environment.

The
results of the ion experiments in this dissertation can be related to the
ion pore theory as previously described. In low calcium aquatic
environments, the ion pores of the surface epithelia would be
submaximally saturated with calcium. This would
lower the force or kinetic energy necessary to strip calcium from the pore.
If environmental calcium were sufficiently low, a rapid and spontaneous flux
of sodium (possibly potassium as well) could occur moving from the fluid of
highest concentration to the fluid of lowest concentration. Diffusion would
be rapid enough that active (energy dependent) uptake or elimination of ions
could not compensate. Death would occur as a result of altered circulatory
volume and/or disrupted ion metabolism. This is consistent with the results
observed in this study as well as the observations of other researchers (as
discussed in the introduction).

----------

As
explained in the introduction and the first pages of discussion, there is
considerable evidence to indicate that the ionic composition of a
teleost's environment directly affects either
gill ionic exchange mechanisms and/or their permeability to certain ions and
water. The results of this dissertation, particularly the ion studies,
appear to substantiate these findings. The concentration of environmental
calcium appears to directly affect survival of red drum in both saltwater
and freshwater environments.

The
diffusional gradients for sodium and calcium
across the surface epithelia of fish placed in low calcium (less than 100
mg/1) sea water (35 g/1 TDS) are in opposite directions. Sodium ions
diffuse from sea water into the extracellular
fluid while calcium would be driven towards sea water. The sharp sodium
gradient causes sodium to influx with high energy. Low calcium
concentrations in the environment would tend to dislodge calcium from its
ion pore binding sites. As calcium begins to flux, the influx of sodium
would become rapid pushing free calcium ions toward the
extracellular fluid. Apparently, the force of
influxing sodium is so great that a minimum concentration of 176 mg/1
calcium (lowest calcium level with survival, Table 7) in sea water is
necessary to begin saturating ion pore binding sites, thus retarding the
influx of sodium and perhaps, that of potassium. Sodium influx and the
concomitant efflux of water are too great in saltwater environments
containing 120 mg/1 calcium or less (treatments with 100% mortality, Table
7). The resultant loss of fluid volume and the increased sodium (and
potassium) content of the extracellular fluid
may cause circulatory shock and cardiac failure (Guyton, 1971), resulting in
death.

In fresh
water, the diffusional gradients for sodium,
potassium and calcium are in the same direction across the surface
epithelia. This unidirectional ion flow, through the ion pores, is the
most reasonable explanation that the euryhaline
red drum can tolerate much lower calcium concentrations in fresh water than
in salt water, 9 mg/1 as opposed to 176 mg/1, respectively. Since calcium
is diffusing in the same direction as sodium and potassium at a relatively
constant concentration, it would keep ion pore binding sites in a state of
comparative saturation, thus retarding ion effluxes. However, when the
environmental concentration falls below a minimum level 1.7 mg/1 (in this
study), the kinetic energy driving calcium and
monovalent ions through the pore would tend to continuously
desaturate calcium binding sites allowing sodium
and potassium (to a lesser extent, calcium) to diffuse into the environment
at a rate greater than active uptake mechanisms could replace them. There
would be a simultaneous water influx. The net effect would be the reduction
in concentration of these ions in the extracellular
fluid. When ionic concentrations reach a critical low level, cardiac spasms
(low ionic tetany) result (Guyton, 1971),
causing death.

If one
uses the concentrations of sodium, potassium and calcium found in saltwater,
vertebrate extracellular fluid, and the minimum
survival treatments of this study and converts them as discussed by Guyton
(1971), one can estimate the osmotic pressure (kinetic energy) driving each
ionic species through the ion pore (Table 23). It is evident from this
table that sodium exerts the greatest force or pressure across cell
membranes (gill epithelia). The ion pore must counteract a sodium pressure
in sea water which is much greater than that in fresh water or low salinity
water. It would seem that the binding affinity for calcium can be altered
in accordance with specific environmental ionic concentrations and total
dissolved solids.

Table 23

A
comparison of osmotic pressure (mmHg) and the direction of diffusion
(flux) for specific ions in fresh and saline waters relative to
extracellular fluid (ECF) at 28ºC

Specific

ions

Osmotic

pressure sea

water: ECF

Ion

flux

Osmotic

pressure1 fresh

water: ECF

Ion

flux

Osmotic

pressure2 saline

water: ECF

Ion

flux

Sodium

6049

influx

2523

efflux

1417

efflux

Potassium

95

influx

93

efflux

67

efflux

Calcium

145

influx

-

-

19

efflux

Low calcium

353a

103b

423c

influx

influx

efflux

384a

474b

-

efflux

efflux

-

-

-

-

-

-

-

1Sodium and potassium
concentrations are those of untreated well water (Table 1).

2Saline water in this
example is sea water (35 g/l) diluted to a strength of 5 g/l (Table
2).

Oduleye (1976) noted that brown trout
pre-adapted to high calcium environments displayed increased salinity
tolerance. The rate of water influx into isolated gills of the thick lipped
mullet could be progressively reduced with adaptation to fresh water (Gallis
et al. , 1979). Low environmental calcium concentrations have been observed
to stimulate prolactin production in
sticklebacks (Wendelaar
Bonga, 1978) and tilapia (WendelaarBonga et al., 1983).
Olesen (1985) demonstrated that serotonin could effect a calcium
related permeability increase in the microvessels
of frog brain. Fleming et al. (1974) indicated that low calcium sea water
stimulated RNA metabolism in the gills of F. kansae.
It is possible that pore binding site affinity for calcium can be altered in
response to osmotic or ionic stress, humoral
factors (e.g. hormones) or messengers to and from individual cell
nucleii. Again, it is important to emphasize
the transient nature of the calcium phenomena detected in the results of
this dissertation. It is unlikely that compensatory mechanisms can be
sufficiently activated over short periods of time (96 hours).

----------

The
results of the freshwater pond culture studies indicated that small red drum
(4-6 g) performed poorly (slow growth and high mortality). Larger red drum
fingerlings (35-45 g) displayed rapid growth and good survival. There are
two reasonable explanations for these observations. Apparently, fish of
greater size underwent less osmotic stress and were not food limited.

It is
generally recognized that larger animals have both a lower surface area to
volume ratio and a lower metabolic rate/unit weight. The gills of fish
represent more than 60% of the exposed body surface (Ogawa, 1975). Large
fish would have a lower surface area to volume ratio than small fish with
respect to their gills as well as body surfaces. In small fish, more
extracellular fluid is brought into close
contact with the environment by way of body surfaces. Therefore, small fish
lose relatively more ions to their environment (fresh water) as a result of
diffusion across leaky permeability barriers. This places a greater demand
on energy dependent mechanisms of ion homeostasis. In addition to osmotic
stress, fish are stressed as a result of handling during stocking. Stress
can effect the release of catecholamines,
corticosteroids and possibly pituitary hormones, all of which can affect
hydromineral balance (Chan et al., 1968; Guyton,
1971; Johnson, 1973; Sage, 1973; Pic et al.,
1974; Shuttleworth, 1978;
Panget al., 1980b; Pang and Yee, 1980; Tomasso
et al., 1980; Robertson, 1984). Hence, small fish are subjected to greater
osmotic stress due to size (surface area) and higher metabolic rates
(greater energy demands). Stress can increase susceptibility to disease,
retard growth and cause death (Stickney, 1979).

Brown, D. J. A. and Lynam, S., 1981. The effect of sodium and
calcium concentrations on the hatching of eggs and the survival of the yolk sac
fry of brown trout, Salmo truttaL. at low pH. J. Fish Biol., 19:205-211.

Chan, D. K. 0., Jones, I. C. and Mosley, W., 1968. Pituitary and
adrenocortical factors in the control of the water and electrolyte composition
of the freshwater European eel (Anguilla anguilla L.).J. Endocr.,
42:91-98.

Ford, P., 1958. Studies on the development of the kidney of the
Pacific pink salmon Oncorhynchus gorbuscha (Walbaum). II. Variation in
glomerular count of the kidney of the Pacific pink salmon. Can. J. Zool.
36:45-47.